Bioresource Technology 133 (2013) 23–30

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Bioresource Technology

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The combined effect of cefazolin and oxytertracycline on biogasproduction from thermophilic anaerobic digestion of dairy manure

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.01.032

⇑ Corresponding author. Tel.: +81 0155495515; fax: +81 0155495519.E-mail address: umetsu@obihiro.ac.jp (K. Umetsu).

Nilmini Beneragama, Suraju A. Lateef, Masahiro Iwasaki, Takaki Yamashiro, Kazutaka Umetsu ⇑Department of Environmental Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan

h i g h l i g h t s

" The effect of cefazolin (CFZ) and oxytetracycline (OTC) on anaerobic digestion was tested." Concentrations of 30, 60 and 90 mg L�1 were compared with no-antibiotic control." CFZ concentrations did not significantly affect (P > 0.05) methane production." Individual OTC and combined CFZ and OTC treatments showed a significant reduction." No noticeable effects on process stability from CFZ and OTC concentrations tested.

a r t i c l e i n f o

Article history:Received 5 October 2012Received in revised form 31 December 2012Accepted 5 January 2013Available online 22 January 2013

Keywords:Anaerobic digestionDairy manureCefazolinOxytetracyclineMethane production

a b s t r a c t

The individual and combined effect of cefazolin (CFZ) and oxytetracycline (OTC) on thermophilic anaer-obic digestion (55 �C) of dairy manure in batch digesters was investigated. Methane productions from theconcentrations tested (30, 60 and 90 mg L�1) were compared with no-antibiotic control. CFZ concentra-tions showed no inhibition (P > 0.05) for methane production. The individual OTC and combined CFZ andOTC at concentrations of 30, 60 and 90 mg L�1 represented 79.1%, 70.3%, 68.6% (P < 0.05) and 88.5%,82.7%, 70.3% (P < 0.05) respectively, of the control values. The high CH4 production, optimal pH andVFA data during digestion indicated the process stability and treatment efficiency.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction mastitis). The use of CFZ as a treatment to mastitis in cows leads

Many antibiotics are being used as veterinary medicines fortherapeutic and sub therapeutic treatments and as feed additivesto promote the growth of animals. Many of these compounds exhi-bit poor retention within the animal gut and intestine during thedigestion, resulting in the excretion of potent parent and daughterproducts (Teeter and Meyerhoff, 2003). Between 17% and 76% ofantibiotics administered to animals are excreted via urine andfeces in an unaltered form or as metabolites of parent compounds(Jjemba, 2002). Oxytetracycline (OTC) and cefazolin (CFZ) are themost commonly used antibiotics in Obihiro University farm, Hok-kaido, Japan. OTC is a broad spectrum antibiotic that is activeagainst variety of bacteria thus used in dairy cattle production pri-marily to treat or prevent diseases and to a lesser extent to increasemilk production or improve feed efficiency. CFZ, a b-lactam antibi-otic, is also widely used to treat bacterial infections in cows (e.g.

antibiotic residues in milk. This milk is not saleable and shouldbe withheld for a period recommended by the manufacturer anddisposed of. The presence of these compounds in manure andwaste milk warrants further consideration due to usage of thesematerials in anaerobic digestion which provides a method ofreducing pollution from agricultural wastes and offers numeroussignificant advantages, such as low sludge production, low energyrequirement, and possible energy recovery (van Staikenburg,1997). The presence of antibiotic compounds has been reportedto affect anaerobic digestion efficiencies, both at the laboratoryand the field scale. Masse et al. (2000) previously reported that tet-racycline and penicillin reduced the methane production of psy-chrophilic anaerobic digestion (20 �C) of swine manure slurry insequencing batch reactor by 25% and 35% respectively. Similarly,Alvarez et al. (2010) reported significant inhibition of anaerobicdigestion of swine manure containing a combination of chlortetra-cycline (CTC) and OTC at concentrations of 10, 50 and 100 mg L�1

at 35 �C, where maximum methane production decreased by 64%in manure containing 100 mg L�1 of both CTC and OTC.

24 N. Beneragama et al. / Bioresource Technology 133 (2013) 23–30

However, there is lack of information on the individual andcombined effect of OTC and CFZ on the anaerobic digestion processof dairy manure at thermophilic temperature (55 �C). It is thereforeof considerable importance to determine whether amending dairymanure with OTC and CFZ (individual and combined) would ad-versely impact process efficiencies and stability of batch digestersused for anaerobic treatment of dairy manure. The manure thatwas directly spiked with environmentally relevant OTC and CFZdoses was used in this study instead of using manure collectedfrom cows administered these antibiotics. Direct OTC and CFZspiked manure enable one to proportionally control the antibioticdoses within each digester.

2. Methods

2.1. Materials

Cow manure and digested slurry were obtained from receptionpit and digester of biogas plant at Obihiro University, Obihiro, Hok-kaido, Japan respectively. Cow manure, discharged to the pit, wasobtained from a herd of lactating Holstein cows and collected dailyfrom concrete floor of free stall barn. The straw (bedding material)presented in manure was separated manually. Digested slurry, theinoculum, is produced from digested cow manure in a digesteroperated at thermophilic temperature (55 �C).

Oxytetracycline (OTC) and cefazolin (CFZ) were obtained fromZigma chemicals.

2.2. Experimental design and procedure

Laboratory-scale anaerobic degradation experiments were per-formed to evaluate the impact of individual OTC and CFZ and com-bined OTC and CFZ presence in manure on biogas production fromthermophilic anaerobic digestion of dairy manure. Three concen-trations; 30, 60, 90 mg L�1 of OTC, CFZ and OTC + CFZ were tested.OTC and CFZ were combined each 50% as to form the same concen-trations (15 + 15, 30 + 30, and 45 + 45 mg L�1). The control and thethree treatments in each group were tested in triplicate in 1 Lbatch digesters with active volume of 700 mL at 55 �C in a thermo-statically controlled water bath for 16 days. For the control andeach treatment, digested slurry (S) and manure (M) were com-bined as to make the ratio slurry 50% and manure 50%. The com-bined contents were thoroughly mixed with a hand mixerseparately. OTC, CFZ and combined OTC and CFZ were addedaccordingly to manure and slurry mixture to obtain the desiredconcentrations of them. 700 mL of each mixture was added to eachdigester in triplicate. Digesters were flushed with argon gas priorto sealing. Gas bags were fixed to each digester to collect theevolved biogas and the digesters were placed in a water bath at55 �C. Digestate samples were taken before and after the experi-ment to analyze for pH, TS (total solids), VS (volatile solids) degra-dation and volatile fatty acids (VFA).

2.3. Analytical methods

Total gas productions were monitored every other day. Wet gasmeter was used to measure the volume of produced gas. All gasmeasurements were expressed at 0 �C and a pressure of one atmo-sphere. Prior to measure the volume of produced gas, gas compo-sition of each gas bag was determined using gas chromatograph(GC) (Shimadzu GC-14A) equipped with a thermal conductivitydetector (stainless column and Porapak Q packing). The opera-tional temperatures of injector port, column and the detector were220, 150 and 220 �C, respectively. Argon was the carrier gas at aflow rate of 50 mL min�1.

TS and VS were measured according to the Standard Methods(Standard Methods, 2005). The pH was measured using HoribaD-55 pH meter. Slurry samples were analyzed for VFA (acetic, pro-pionic, butyric and formic acids) with a high performance liquidchromatograph (HPLC, Shimadzu LC-10AD) with Shim-Pack SCR-102H column. The analytical procedure used in the current studyfor analyzing VFA using HPLC was described in detail by Kimuraet al. (1994).

2.4. Data analysis

The statistical analyses were performed using a two-sample t-test assuming unequal variances from the statistical software SAS9.2. Significance was accepted at probabilities (p) of 0.05 or lessfor all analysis.

The lag phase for methane production in the anaerobic digesterwas analyzed using a Gompertz model as shown below.

Mp ¼ Pm � exp �expRm

Pm

� �ðk� tÞeþ 1

� �� �ð1Þ

where Mp is the cumulative methane production (mL), Pm is themethane production potential (mL), Rm is methane production rate(mL/day), k is lag-phase time (d), e is exponential 1 and t is time.

3. Results and discussion

3.1. Performance of anaerobic treatment of dairy manure with andwithout cefazolin

3.1.1. Methane productionMethane and carbon dioxide are the terminal end-products of

dairy manure anaerobic digestion. Hence, the methane volume inthe produced biogas is a better indicator of digester performance.Methane production per g of VS added for each batch treatmentof control and cefazolin spiked (30, 60 and 90 mg L�1), in everyother day is presented in Fig. 1(a). Fig. 1(b) shows the cumulativemethane production during the period of digestion (16 days).

Methane production was observed from digesters of no-cefazo-lin control (SM) and cefazolin spiked at concentration of 30 mg L�1

(SM(C30)) with no lag phase and other two digesters with cefazolinconcentration of 60 mg L�1 (SM(C60)) and 90 mg L�1 (SM(C90))with a lag phase of about one day. SM and SM(C30) digestersreached peak methane production (38.49 and 35.71 mL/gVS addedrespectively) by the fourth day of digestion while SM(C60) andSM(C90) reached their peak (36.82 and 35.03 mL/gVS addedrespectively) by sixth day of digestion due to the initial lag phase.Methanogens are responsible for producing methane in an anaero-bic digestion process. As the anaerobic digestion process advancesand the methanogen populations become more established, theproduction of methane begins to increase. However, cefazolin con-centrations of 60 and 90 mg L�1 have exerted some pressure onmethanogens at the beginning so that methanogens had to under-go some acclimatization period and as a result, a lag phase could beobserved in those two digesters. After 16 days, the mean cumula-tive total biogas production values for digesters SM, (SM(C30)),(SM(C60)) and (SM(C90)) were 10.544 ± 0.329, 10.5 ± 0.497,10.693 ± 0.088 and 11.065 ± 0.574 L respectively.

The methane productivity of SM, (SM(C30)), (SM(C60)) and(SM(C90)) in terms of volatile solids added (mL/g VS added) were145.026 ± 3.171, 148.142 ± 7.051, 150.482 ± 3.079 and159.253 ± 5.952 mL respectively. The no-antimicrobial controltreatment (SM) showed no significant difference (P > 0.05) inmethane volume with the (SM(C30)), (SM(C60)) and (SM(C90)).Although a decreasing trend in methane production was expectedas the concentration of cefazolin was increased, it showed a pecu-

Fig. 1. Daily (a) and cumulative (b) methane gas volume obtained from the control (SM) and the digesters spiked with cefazolin at concentrations of 30 mg L�1 (SM(C30)),60 mg L�1 (SM(C60)) and 90 mg L�1 (SM(C90)) during the digestion period (16 days).

N. Beneragama et al. / Bioresource Technology 133 (2013) 23–30 25

liar result leading to believe that the cefazolin concentrationstested would not exert any inhibitory effect on total and methanegas productions during anaerobic digestion of dairy manure. Theprevious experiment conducted with added 10 mg L�1 cefazolininto the substrates (data are not shown) has also confirmed thatthe tested concentration would not effect on total and methanegas productions during anaerobic digestion of dairy manure andwaste milk. However, SM(C60) and SM(C90) showed a short lagphase of 0.4 and 0.5 d respectively for methane production. Theproduced cumulative methane volumes each other day comparedbetween SM(C30) and SM(C60), SM(C30) and SM(C90) andSM(C60) and SM(C90) showed non-significant difference(P > 0.05). This clearly evidences that there is hardly any inhibitoryeffect exerted on biogas production at these concentrations ofcefazolin. Although many researches have shown the effect of dif-ferent antibiotics in different concentrations on bio gas production,no any study regarding cefazolin could be found in the literature.Poels et al. (1984) have reported a decreased methane productionin the presence of antibiotics thiamphenicol and amoxicillin as aresult of accumulation of VFAs. Similarly Masse et al. (2000) have

shown a 25% reduction of methane generation by the presence oftetracycline in swine manure sequencing batch reactors. Further,Sanz et al. (1996) found chlortetracycline (CTC) to be a powerfulinhibitor of anaerobic digestion and concluded that methane inhi-bition was due to accumulation of acetogenic substrates. The non-significant differences in methane production between cefazolinand no-cefazolin treatments in our experiment are best explainedby the adaptation of methanogenic population to cefazolin antibi-otic or the methanogenic bacteria present in the waste have al-ready been resistant to cefazolin. This could probably be thereason since cefazolin is mainly being used to treat mastitis incows at Obihiro University farm, hence causing to spread the cefaz-olin resistant bacteria in the vicinity of the environment.

3.1.2. pHThe initial and final pH data are shown in Fig. 2. The initial pH

for all reactors ranged between 7.47 and 7.65 which are consideredsuitable pH for anaerobic digestion. After 16 days of digestion, pHreached a range between 7.70 and 7.75 suggesting that the organicacid concentration was balanced with the fermentative and meth-

Fig. 2. pH change of the digesters SM, SM(C30), SM(C60), SM(C90), SM(T30), SM(T60), SM(T90), SM(CT30), SM(CT60), SM(CT90) over the digestion period of 16 days.

26 N. Beneragama et al. / Bioresource Technology 133 (2013) 23–30

anogenic microbial activities. Typically, a sharp decrease in pHwould suggest reduced methanogenic activity and subsequentVFA accumulation. Nevertheless, all reactors of current experimentappeared to be sufficiently buffered to minimize pH fluctuationsand have undergone digestion without a failure.

3.1.3. Volatile solids (VS) consumptionTotal solids (TS) and volatile solids (VS) concentrations before

and after the experiment remained relatively constant for theSM, SM(C30), SM(C60) and SM(90) treatments. The values werenot significantly different (P > 0.05) between each treatment.Moreover, a non-significantly different (P > 0.05) VS consumptionvalues of 24.06%, 21.98%, 26.77% and 24.47% could be observedfor SM, SM(C30), SM(C60) and SM(C90) respectively.

3.1.4. Volatile fatty acids (VFA)Analysis of VFAs of samples from each digester was performed

prior to the digestion. Since we used the same materials for prepar-ing the substrates for digestion, it showed an almost similar con-centration of VFAs in all the digester contents regardless of theantibiotic concentrations spiked. Upon completion of 16 day diges-tion, low concentrations of all VFAs assayed were observed in allthe digesters (Fig. 3). At the end of digestion, total VFA concentra-tions of 152.07 ± 57.48, 321.71 ± 50.93, 225.45 ± 16.81 and249.77 ± 77.68 mg L�1 (P > 0.05) in SM, SM(C30), SM(C60) andSM(C90) respectively were observed. Formate was not detectedin any slurry mixture, while propionate presented at higher con-centration (232.43 ± 44.36 mg L�1) in SM(C30) than other digest-ers. No accumulation of VFA could be seen in either digesterconfirming the process stability.

These data support the non-significant difference in gas produc-tion between cefazolin spiked and no-cefazolin control treatments.Further, it also reveals that the digestion process in each digesterhas undergone without a failure.

3.2. Performance of anaerobic treatment of dairy manure with andwithout oxytetracycline

3.2.1. Methane productionDaily and cumulative methane production values from digest-

ers containing oxytetracycline (OTC) concentrations of 30 mg L�1

(SM(T30)), 60 mg L�1 (SM(T60)), 90 mg L�1 (SM(T90)) and no-OTC control (SM) are shown in Fig. 4.

Similar to the methane production observed in the digester con-taining cefazolin concentration 30 mg L�1, the digesters with dif-ferent OTC concentrations did not show any initial lag phase ofmethane generation. Maximum methane production was achievedat fourth day of digestion in all digesters except SM(T30) whichgave the peak at second day of digestion. The cumulative biogasproduction after 16 days of digestion of SM(T30), SM(T60) andSM(T90) were 8.262 ± 0.477, 7.708 ± 0.540 and 8.018 ± 0.670 Lrespectively. These values were significantly different (P < 0.05)from the cumulative biogas production (10.544 ± 0.329 L) of SM.However, no significant difference (P > 0.05) in cumulative biogasproduction could be observed among SM(T30), SM(T60) andSM(T90).

Similar trend was observed in cumulative methane production.Methane production increased for all treatments during digestionperiod, however a significantly (P < 0.05) low methane productionwas observed in all the treatments with OTC, each showing no sig-nificant difference (P > 0.05) with other OTC treatments. In fact, themethane production in SM(T30), SM(T60) and SM(T90) repre-sented 79.1%, 70.3% and 68.6% respectively, of the control valuesat the end of 16 day digestion period. This clearly gives the evi-dence of OTC causing a lower but significant inhibitory effect at acertain concentration above which it does not cause any significanthindrance to methanogenic activity. None of the concentrationstested showed a lag phase for methane production.

Similar findings have been reported in the literature for differ-ent wastes. Lallai et al. (2002) in a study conducted to find the ef-fect of OTC on biogas production in the anaerobic digestion of pigwaste slurry, found minimal differences in methane production be-tween OTC (range of concentrations studied were 125 and250 mg L�1) and no-antimicrobial treatments, and concluded thatboth the acid-forming and methane-forming bacteria were not af-fected by the presence of OTC. Similar results in testing its effectson the anaerobic digestion of cow manure have been obtained byGamel-El-Din (1986). Arikan et al. (2006) have also produced somecomparable results in which they have found a 27% lower (P < 0.05)cumulative biogas production from digesters containing manurefrom medicated calves with OTC relative to that from digesterscontaining un-medicated manure. The reported OTC level in themanure used in the study was 9.8 ± 0.1 mg L�1. Although the con-centration of OTC is seemed to be lower than the levels used in ourstudy, it still showed some inhibitory effect on biogas production.However, there were no significant differences in biogas methanecontent. We could observe some variations in the results from dif-

Fig. 3. Volatile fatty acids (VFA) concentration of substrates of each digester before (a) and after (b) the digestion.

N. Beneragama et al. / Bioresource Technology 133 (2013) 23–30 27

ferent studies (Masse et al., 2000; Fedler and Day, 1985). These arelikely due to differences in antibiotic concentration, manuresource, reactor size, inoculum manure ratio, feeding method (batchor continuous) and source of inoculum.

3.2.2. pHThe pH value data before and after the experiment are shown in

Fig. 2. At the beginning, the pH values of SM, SM(T30), SM(T60) andSM(T90) ranged between 7.47 and 7.63. A slight increase in pH val-ues were observed in all digesters ranging from 7.57 to 7.74 at theend of digestion. The presence of OTC in manure showed no effecton pH during digestion. An optimal pH range of 6.7–7.4 has beenreported for most methanogenic bacteria and the rate of methano-genesis may decrease if the pH is <6.3 or >7.8. pH values of alldigesters increased towards the end of digestion remaining below7.8. This suggests that the process stability of all the digesters hasbeen maintained without a failure.

3.2.3. VS consumptionVS consumption percentages of 24.06%, 17.99%, 17.31% and

18.97% for SM, SM(T30), SM(T60) and SM(T90) respectively sup-port the methane production behavior among SM, SM(T30),

SM(T60) and SM(T90). Significantly lower VS consumption per-centage in SM(T30), SM(T60) and SM(T90) than SM reveals theinhibition of methane production with spiked OTC levels.

3.2.4. VFAVFA assay data showed the presence of formate, acetate, propi-

onate, and butyrate in all the slurry mixtures (Fig. 3). All treat-ments (SM(T30), SM(T60) and SM(T90)) exhibited an increase intotal VFAs compared to the control (SM) with acetate comprisingof 52.25%, 68.05% and 75.83% of all VFA in SM(T30), SM(T60) andSM(T90) respectively. Similarly, Stone et al. (2009) reported signif-icant VFA accumulation for CTC-containing manure under batchanaerobic experimental conditions. VFA accumulation was thoughtdue to subsequent inhibition of methanogenic activities (Masseet al., 2000).

3.3. Performance of anaerobic treatment of dairy manure with andwithout combination of cefazolin and oxytetracycline

3.3.1. Methane productionThe combined effect of cefazolin and oxytetracycline at concen-

trations of 0 mg L�1 (SM), 30 mg L�1 (SM(CT30)), 60 mg L�1

Fig. 4. Daily (a) and cumulative (b) methane gas volume obtained from the control (SM) and the digesters spiked with oxytetracycline at concentrations of 30 mg L�1

(SM(T30)), 60 mg L�1 (SM(T60)) and 90 mg L�1 (SM(T90)) during the digestion period (16 days).

28 N. Beneragama et al. / Bioresource Technology 133 (2013) 23–30

(SM(CT60)) and 90 mg L�1 (SM(CT90)) on methane production arepresented in Fig. 5.

Fig. 5(a) shows the daily methane production volume per gramVS added (mL/g VS added) in each four digesters. The peak meth-ane production was observed at fourth day in all digesters. Cumu-lative methane yields of SM(CT30), SM(CT60) and SM(CT90)showed a significant difference (P < 0.05) with the cumulativemethane yield of SM (Fig. 5(b)). This behavior was similar to thedigesters spiked with OTC. However, no significant differences ofcumulative methane yields could be observed between digestersspiked with combined CFZ and OTC in different concentrations.Cumulative methane production from SM(CT30), SM(CT60) andSM(CT90) was 88.5%, 82.7% and 70.3% respectively of what was ob-served from SM. No lag phase for methane production was shownby either digester.

3.3.2. pHThe spiking of manure with CFZ and OTC showed no effect on

pH during digestion. The initial pH values for SM(CT30), SM(CT60)and SM(CT90) were 7.54, 7.52 and 7.56, respectively. pH values in-

creased in all the digesters during 16 day digestion and final pHvalues of 7.7, 7.69 and 7.59 respectively were observed (Fig. 2).These pH values lie within the optimal range for most methano-genic bacteria, hence a digestion failure could not be observed.

3.3.3. VS consumptionVolatile solids of the samples calculated before and after the

digestion resulted a VS consumption percentage values of 24.06%,19.58%, 13.53% and 15.1% for SM, SM(CT30), SM(CT60) andSM(CT90) respectively. Significantly different (P < 0.05) VS con-sumption values in SM(CT30), SM(CT60) and SM(CT90) to the SMconfirm the hindrance of methanogenesis by incorporating bothCFZ and OTC in respective concentrations. Nevertheless, it didnot show any significant inhibition with increasing the concentra-tion of CFZ and OTC from 30 to 90 mg L�1.

3.3.4. VFAFinal VFA concentrations of SM(CT30), SM(CT60) and SM(CT90)

showed an increasing total VFA concentration with increasing anti-biotic concentration (Fig. 3). Total VFA values of 208.17 ± 33.16,

Fig. 5. Daily (a) and cumulative (b) methane gas volume obtained from the control (SM) and the digesters spiked with combined cefazolin and oxytetracycline atconcentrations of 30 mg L�1 (SM(CT30)), 60 mg L�1 (SM(CT60)) and 90 mg L�1 (SM(CT90)) during the digestion period (16 days).

N. Beneragama et al. / Bioresource Technology 133 (2013) 23–30 29

346.42 ± 21.55 and 918.24 ± 6.74 mg L�1 comprising of acetateconcentrations of 97.51 ± 7.11, 253.19 ± 21.92 and674.80 ± 8.83 mg L�1 were obtained for SM(CT30), SM(CT60) andSM(CT90) respectively. Although the methane productions ofSM(CT30), SM(CT60) and SM(CT90) were non-significantly differ-ent, a considerable reduction could be observed in SM(CT90) thanSM(CT30) and SM(CT60) (Fig. 5). These results are comparable withfinal VFA concentrations of each digester. SM(CT90) showing aconsiderable higher final VFA value (918.24 ± 6.74 mg L�1) thanSM(CT30) and SM(CT60), supports the methane production data.

Similar findings have been reported by Poels et al. (1984). Theyhave reported an accumulation of VFAs resulting in decreasedmethane production in the presence of antibiotics thiamphenicoland amoxicillin. In the current study, we present some new results.The presence of CFZ in manure in used concentrations showed nosignificant effect on biogas and methane production. However, alag phase of methane production was observed with higher CFZconcentrations. The same trend could be seen with spiked OTCconcentrations in manure though the methane and biogas produc-

tion from manure spiked with different OTC concentrationsshowed a significantly (P < 0.05) lower values than the no-OTCcontrol. Similar results were observed with manure spiked withboth antibiotics.

The comparison results among the same concentration of twoantibiotics and the combination of the two showed almost similargas production in SM(T30) and SM(CT30). Having no significantdifference with no-antibiotic control, SM(C30) treatment resultedin a significantly higher methane production compared toSM(T30) and SM(CT30). Methane production of SM(C60) andSM(C90) compared with SM(T60),SM(CT60) and SM(T90),SM(CT90) respectively also followed the similar pattern. Therefore,in relation to the antibiotics used, cefazolin in used concentrationsseemed not to exert any effect on the methane production whereasoxytetracycline alone and its combination with cefazolin in usedconcentrations seemed to have significant effect. OTC alone andits combination with CFZ seemed to exert the similar suppressingeffect on methane production albeit either one did not show anyincreasing effect with increasing concentration in manure. These

30 N. Beneragama et al. / Bioresource Technology 133 (2013) 23–30

findings suggest that methanogenic inhibition appears likely dueto the presence of high concentrations of individual OTC and com-bination of CFZ and OTC, and consequently, caused an increase inVFA specially acetic acid in the digesters. Moreover, althoughmethane production was lower in digesters fed with individualOTC and OTC combined with CFZ, the treatment efficiency wasnot seemed to be affected. Final pH of all the digesters remainedwith minimal differences.

The effect of adding OTC to the manure has also been shown bySankvist et al. (1984). They reported that in a semi-continuous flowthermophilic fermenter with a hydraulic retention time of 5–7 days, methane production was reduced by 50% after six consecu-tive days of adding OTC at a rate of 100 mg L�1 of manure. The ob-served non-significant 11.46%, 17.3% and 29.75% (P > 0.05)methane reduction percentages of SM(T30), SM(T60) andSM(T90) of our study confirm the results obtained by Sankvistet al. (1984). However, a study conducted by Fedler and Day(1985) showed although manure from pigs fed 100 mg chlortetra-cycline (CTC)/kg of feed reduced methane production by about20%, when CTC was added directly into the fermenter, there wasno reduction in methane production. As suggested by Sankvistet al. (1984), this could be due to the metabolites of CTC producedin the gastrointestinal tract of the animal. For this reason, furtherexperimentation with manure from cows fed with each antibioticshould be considered.

It is also important to discuss about the effect of temperature ondegradation of CFZ and OTC during thermophilic anaerobic diges-tion in order to resolve whether it has an effect on the different re-sults obtained between CFZ and OTC of the current study. The fateof OTC during the anaerobic digestion of manure at 35 �C has beenstudied by Arikan et al. (2006) and reported a 60% removal of OTCin 64 days yielding a calculated half-life of 56 days. No studies fo-cused on the degradation of OTC at 55 �C and of CFZ at 35 or 55 �Cwere available. Hence, further experimentation in this regard issuggested.

4. Conclusion

The individual and combined effects of CFZ and OTC on anaero-bic digestion of dairy manure were examined in this study. Cefaz-olin did not have any inhibitory effect on methane production at

the concentrations tested. However, the inhibition exerted by indi-vidual OTC and combined CFZ and OTC at used concentrations (30,60, 90 mg L�1) on methane yield followed almost a similar patternwith no significant differences between respective antimicrobialconcentrations of both treatments. The high CH4 production, pHand VFA data obtained during digestion indicated that the antibiot-ics did not have any negative effects on process stability.

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